High-purity ssRNA molecular weight marker as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of molecular detection, and discloses a high-purity ssRNA molecular weight marker as well as a preparation method and application thereof. According to the preparation method of the ssRNA molecular weight marker provided by the invention, a forward primer comprising a T7 promoter fragment and a reverse primer comprising a polythymine fragment with the length of 20-150nt are creatively introduced in a PCR (Polymerase Chain Reaction) amplification process, so that an obtained DNA (Deoxyribonucleic Acid) template can be subjected to in-vitro transcription under the action of T7 RNA polymerase, thereby obtaining an IVT (Interleukinine Vitamin Transferase) stock solution containing ssRNA; at the moment, only ssRNA in the IVT stock solution has free polyadenine fragments, so that no additional DNA hydrolase needs to be introduced, the high-purity ssRNA molecular weight marker can be collected and purified only through simple oligo (dT) enrichment treatment, and the ssRNA molecular weight marker has good stability; the method effectively solves the problems of complex operation, low product purity and unstable product in the preparation of the existing ssRNA molecular weight marker, and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular detection, and in particular relates to a high-purity ssRNA molecular weight marker and a preparation method and application thereof. Background Art

[0002] Single-stranded RNA (ssRNA) refers to an RNA molecule composed of a single nucleotide chain, which has multiple functions in organisms, such as transmitting genetic information as messenger RNA (mRNA), participating in protein synthesis as ribosomal RNA (rRNA), helping to transport amino acids to the site of protein synthesis as transfer RNA (tRNA), and serving as genetic material in some viruses. The molecular weight of ssRNA determines its functions and characteristics in various biological processes, so the accurate determination of ssRNA molecular weight is of great significance for studies such as gene expression regulation, viral infection and RNA interference.

[0003] ssRNA molecular weight markers refer to mixed reagents including a series of ssRNA molecules of different molecular weights, which are used to calibrate the molecular weight of the ssRNA fragments to be measured. In the process of preparing ssRNA molecular weight standards, the ssRNA structure is unstable and easily interfered by various external factors, causing denaturation and degradation. Each RNA may combine with each other to form a complex structure of dsRNA, and is very sensitive to degradation by additional exogenous ribonucleases. There are problems such as complex operation, low product purity and product instability, which have great limitations. Summary of the invention

[0004] The first object of the present invention is to solve the problems of low product purity and instability in the preparation of existing ssRNA molecular weight markers, and to provide a method for preparing ssRNA molecular weight markers.

[0005] The second object of the present invention is to provide a ssRNA molecular weight marker.

[0006] The third object of the present invention is to provide the use of the above-mentioned ssRNA molecular weight marker in molecular detection.

[0007] Specifically, the preparation method of the ssRNA molecular weight marker provided by the present invention specifically comprises: taking a primer composition to perform PCR amplification on a plasmid template to obtain a DNA template; taking the DNA template to perform in vitro transcription under the action of T7 RNA polymerase to obtain an IVT stock solution; taking the IVT stock solution to perform oligo (dT) enrichment treatment to obtain the ssRNA molecular weight marker; wherein the primer composition comprises a forward primer and a reverse primer, the forward primer comprises a T7 promoter fragment, and the reverse primer comprises a 20-150 nt polythymine fragment.

[0008] Furthermore, the nucleotide sequence of the T7 promoter fragment is specifically: TAATACGACTCACTATA.

[0009] Further, when the plasmid template includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 1, the primer composition includes a forward primer with a sequence as shown in SEQ ID NO: 2 and a reverse primer with a sequence as shown in SEQ ID NO: 3, and the length of the ssRNA molecular weight marker is 6000 nt.

[0010] Further, when the plasmid template includes a nucleotide fragment with a sequence as shown in SEQ ID NO:4, the primer composition includes a forward primer with a sequence as shown in SEQ ID NO:5 and a reverse primer as described in SEQ ID NO:6, and the length of the ssRNA molecular weight marker is 4000 nt.

[0011] Further, when the plasmid template includes a nucleotide fragment with a sequence as shown in SEQ ID NO:7, the primer composition includes a forward primer with a sequence as shown in SEQ ID NO:8 and a reverse primer as described in SEQ ID NO:9, and the length of the ssRNA molecular weight marker is 3000 nt.

[0012] Further, when the plasmid template includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 10, the primer composition includes a forward primer with a sequence as shown in SEQ ID NO: 11 and a reverse primer as shown in SEQ ID NO: 12, and the length of the ssRNA molecular weight marker is 2000 nt.

[0013] Further, when the plasmid template includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 13, the primer composition includes a forward primer with a sequence as shown in SEQ ID NO: 14 and a reverse primer as described in SEQ ID NO: 15, and the length of the ssRNA molecular weight marker is 1500 nt.

[0014] Further, when the plasmid template includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 16, the primer composition includes a forward primer with a sequence as shown in SEQ ID NO: 17 and a reverse primer as shown in SEQ ID NO: 18, and the length of the ssRNA molecular weight marker is 1000 nt.

[0015] Further, when the plasmid template includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 19, the primer composition includes a forward primer with a sequence as shown in SEQ ID NO: 20 and a reverse primer as shown in SEQ ID NO: 21, and the length of the ssRNA molecular weight marker is 500 nt.

[0016] Further, when the plasmid template includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 22, the primer composition includes a forward primer with a sequence as shown in SEQ ID NO: 23 and a reverse primer as shown in SEQ ID NO: 24, and the length of the ssRNA molecular weight marker is 200 nt.

[0017] Furthermore, the PCR amplification reaction system includes: Taq enzyme, PCR buffer, dNTP, forward primer, reverse primer and plasmid template.

[0018] Furthermore, the reaction conditions of the PCR amplification include: pre-denaturation at 92-98°C for 1-5 min; denaturation at 92-98°C for 25-35 s, annealing at 60-65°C for 3-10 s, extension at 70-75°C for 65-75 s, 30-40 cycles; 70-75°C for 3-7 min.

[0019] Furthermore, the in vitro transcription reaction system includes: RNA polymerase, inorganic pyrophosphatase, RNase inhibitor, dNTP, transcription buffer and DNA template.

[0020] Furthermore, the temperature of the in vitro transcription is 35-40° C., and the time is 1-10 hours.

[0021] Furthermore, the oligo(dT) enrichment treatment includes one or more of oligo(dT) chromatography enrichment treatment, oligo(dT) magnetic bead enrichment treatment and oligo(dT) centrifugal column enrichment treatment.

[0022] The ssRNA molecular weight marker provided by the present invention is prepared by the above-mentioned method for preparing the ssRNA molecular weight marker.

[0023] Among them, the nucleotide sequence of the ssRNA6000 fragment is shown in SEQ ID NO:25, the nucleotide sequence of the ssRNA4000 fragment is shown in SEQ ID NO:26, the nucleotide sequence of the ssRNA3000 fragment is shown in SEQ ID NO:27, the nucleotide sequence of the ssRNA2000 fragment is shown in SEQ ID NO:28, the nucleotide sequence of the ssRNA1500 fragment is shown in SEQ ID NO:29, the nucleotide sequence of the ssRNA1000 fragment is shown in SEQ ID NO:30, the nucleotide sequence of the ssRNA500 fragment is shown in SEQ ID NO:31, and the nucleotide sequence of the ssRNA200 fragment is shown in SEQ ID NO:32.

[0024] The present invention also provides the use of the above ssRNA molecular weight marker in molecular detection.

[0025] Furthermore, the application includes: taking the ssRNA molecular weight marker for denaturation treatment to obtain a denatured product; taking the denatured product as an RNA marker to determine the molecular weight of a sample to be tested.

[0026] Furthermore, the heat treatment comprises: incubating the ssRNA molecular weight marker at 65-70° C. for 8-10 minutes, and then cooling at 0-2° C. for 3-5 minutes to obtain the heat treatment product.

[0027] Beneficial effects:

[0028] In the preparation method of the ssRNA molecular marker provided by the present invention, a forward primer including a T7 promoter fragment and a reverse primer including a polythymine fragment with a length of 20 to 150 nt are creatively introduced during the PCR amplification process, so that the obtained DNA template can be transcribed in vitro under the action of T7 RNA polymerase, thereby obtaining an IVT stock solution containing ssRNA, and at this time, only the ssRNA in the IVT stock solution has a free polyadenine fragment, so there is no need to introduce additional DNA hydrolase, and only a simple oligo (dT) enrichment treatment is required to collect and purify the high-purity ssRNA molecular weight marker, and the ssRNA molecular weight marker has good stability; the problems of complex operation, low product purity and product instability in the preparation of existing ssRNA molecular weight markers are effectively solved, and the method has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 A gel electrophoresis diagram of a DNA template provided in an embodiment of the present invention;

[0030] Figure 2 The gel electrophoresis diagram of the IVT stock solution provided in the embodiment of the present invention;

[0031] Figure 3 This is a gel electrophoresis diagram of the ssRNA molecular weight markers provided in the embodiments of the present invention. DETAILED DESCRIPTION

[0032] The nucleotide sequences involved in the present invention are specifically shown in Table 1.

[0033] Table 1.

[0034]

[0035]

[0036]

[0037]

[0038]

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055] The preparation method of the ssRNA molecular weight marker provided by the present invention specifically comprises: taking a primer composition to perform PCR amplification on a plasmid template to obtain a DNA template; taking the DNA template to perform in vitro transcription under the action of T7 RNA polymerase to obtain an IVT stock solution; taking the IVT stock solution to perform oligo (dT) enrichment treatment to obtain the ssRNA molecular weight marker.

[0056] In the present invention, the primer composition includes a forward primer and a reverse primer. The forward primer includes a T7 promoter fragment, and the nucleotide sequence of the T7 promoter fragment is specifically: TAATACGACTCACTATA. The reverse primer includes a polythymine fragment, and the length of the polythymine fragment is 20 to 150 nt, such as 20nt, 23nt, 28nt, 30nt, 40nt, 50nt, 100nt, 150nt or any value therebetween.

[0057] In the present invention, the plasmid template specifically includes one or more of a nucleotide fragment with a sequence as shown in SEQ ID NO: 1, a nucleotide fragment with a sequence as shown in SEQ ID NO: 4, a nucleotide fragment with a sequence as shown in SEQ ID NO: 7, a nucleotide fragment with a sequence as shown in SEQ ID NO: 10, a nucleotide fragment with a sequence as shown in SEQ ID NO: 13, a nucleotide fragment with a sequence as shown in SEQ ID NO: 16, a nucleotide fragment with a sequence as shown in SEQ ID NO: 19, and a nucleotide fragment with a sequence as shown in SEQ ID NO: 22.

[0058] In the present invention, based on the above-mentioned plasmid template, the inventors take the introduction of a T7 promoter fragment in the forward primer and a polythymine fragment in the reverse primer as the basic purpose, and take improving the binding performance of the forward primer, the reverse primer and the plasmid template and improving the chain extension efficiency as further improvement goals, and creatively design a primer combination for each plasmid template. Although the forward primer and the reverse primer in the primer combination introduce the T7 promoter fragment and the polythymine fragment with non-complementary parts, using them to perform PCR amplification on the corresponding plasmid template still has excellent amplification effect.

[0059] In some specific embodiments, when the plasmid template includes a nucleotide fragment with a sequence as described in SEQ ID NO: 1, the primer composition preferably includes a forward primer with a sequence as described in SEQ ID NO: 2 and a reverse primer with a sequence as described in SEQ ID NO: 3, and the length of the ssRNA molecular weight marker is 6000 nt.

[0060] In some specific embodiments, when the plasmid template includes a nucleotide fragment with a sequence as shown in SEQ ID NO:4, the primer composition preferably includes a forward primer with a sequence as shown in SEQ ID NO:5 and a reverse primer as shown in SEQ ID NO:6, and the length of the ssRNA molecular weight marker is 4000 nt.

[0061] In some specific embodiments, when the plasmid template includes a nucleotide fragment with a sequence as shown in SEQ ID NO:7, the primer composition preferably includes a forward primer with a sequence as shown in SEQ ID NO:8 and a reverse primer as shown in SEQ ID NO:9, and the length of the ssRNA molecular weight marker is 3000 nt.

[0062] In some specific embodiments, when the plasmid template includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 10, the primer composition preferably includes a forward primer with a sequence as shown in SEQ ID NO: 11 and a reverse primer as shown in SEQ ID NO: 12, and the length of the ssRNA molecular weight marker is 2000 nt.

[0063] In some specific embodiments, when the plasmid template includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 13, the primer composition preferably includes a forward primer with a sequence as shown in SEQ ID NO: 14 and a reverse primer as shown in SEQ ID NO: 15, and the length of the ssRNA molecular weight marker is 1500 nt.

[0064] In some specific embodiments, when the plasmid template includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 16, the primer composition preferably includes a forward primer with a sequence as shown in SEQ ID NO: 17 and a reverse primer as shown in SEQ ID NO: 18, and the length of the ssRNA molecular weight marker is 1000 nt.

[0065] In some specific embodiments, when the plasmid template includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 19, the primer composition preferably includes a forward primer with a sequence as shown in SEQ ID NO: 20 and a reverse primer as shown in SEQ ID NO: 21, and the length of the ssRNA molecular weight marker is 500 nt.

[0066] In some specific embodiments, when the plasmid template includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 22, the primer composition preferably includes a forward primer with a sequence as shown in SEQ ID NO: 23 and a reverse primer as shown in SEQ ID NO: 24, and the length of the ssRNA molecular weight marker is 200 nt.

[0067] In the present invention, based on the above-mentioned plasmid template and primer composition, the DNA template obtained by PCR amplification specifically includes one or more of the nucleotide fragments shown in SEQ ID NO: 1, the nucleotide fragments shown in SEQ ID NO: 4, the nucleotide fragments shown in SEQ ID NO: 7, the nucleotide fragments shown in SEQ ID NO: 10, the nucleotide fragments shown in SEQ ID NO: 13, the nucleotide fragments shown in SEQ ID NO: 16, the nucleotide fragments shown in SEQ ID NO: 19 and the nucleotide fragments shown in SEQ ID NO: 22.

[0068] In the present invention, the PCR amplification is a technical means commonly used in the field of molecular biology. Those skilled in the art can make adaptive choices according to actual needs, and the present invention does not impose any special limitation on it.

[0069] In some specific embodiments, the PCR amplification reaction system preferably includes: Taq enzyme, PCR buffer, dNTP, forward primer, reverse primer and plasmid template.

[0070] In some specific embodiments, the reaction conditions of the PCR amplification preferably include: pre-denaturation at 92-98°C for 1-5 min; denaturation at 92-98°C for 25-35 s, annealing at 60-65°C for 3-10 s, extension at 70-75°C for 65-75 s, 30-40 cycles; 70-75°C for 3-7 min.

[0071] In the present invention, the in vitro transcription is a technical means commonly used in the field of molecular biology. Those skilled in the art can make adaptive choices according to actual needs, and the present invention does not impose any particular limitation on it.

[0072] In some specific embodiments, the in vitro transcription reaction system preferably includes: RNA polymerase, inorganic pyrophosphatase, RNase inhibitor, dNTP, transcription buffer and DNA template.

[0073] In some specific embodiments, the in vitro transcription conditions include a temperature of preferably 35-40°C, such as 35°C, 37°C, 37.5°C, 39°C, 40°C or any value therebetween; and a time of preferably 1-10h, such as 1h, 3h, 5h, 7h, 9h, 10h or any value therebetween.

[0074] In the present invention, specific examples of the oligo(dT) enrichment treatment include but are not limited to: one or more of oligo(dT) chromatography enrichment treatment, oligo(dT) magnetic bead enrichment treatment and oligo(dT) centrifugal column enrichment treatment.

[0075] Based on the above-mentioned method for preparing ssRNA molecular weight markers, the present invention further provides a ssRNA molecular weight marker. The ssRNA molecular weight marker is prepared by the above-mentioned method for preparing ssRNA molecular weight markers.

[0076] In some specific embodiments, the ssRNA molecular weight marker preferably includes one or more of RNA6000 fragment, RNA4000 fragment, RNA3000 fragment, RNA2000 fragment, RNA1500 fragment, RNA1000 fragment, RNA500 fragment and RNA200 fragment. The nucleotide sequence of the RNA6000 fragment is shown in SEQ ID NO:25, the nucleotide sequence of the RNA4000 fragment is shown in SEQ ID NO:26, the nucleotide sequence of the RNA3000 fragment is shown in SEQ ID NO:27, the nucleotide sequence of the RNA2000 fragment is shown in SEQ ID NO:28, the nucleotide sequence of the RNA1500 fragment is shown in SEQ ID NO:29, the nucleotide sequence of the RNA1000 fragment is shown in SEQ ID NO:30, the nucleotide sequence of the RNA500 fragment is shown in SEQ ID NO:31, and the nucleotide sequence of the RNA200 fragment is shown in SEQ ID NO:32.

[0077] Based on the above ssRNA molecular weight marker, the present invention also provides its application in molecular detection.

[0078] In the invention, the application of the ssRNA molecular weight marker in molecular detection specifically includes: taking the ssRNA molecular weight marker for heat treatment to obtain a heat treatment product; taking the heat treatment product as an RNA marker to determine the molecular weight of the sample to be tested.

[0079] In some specific embodiments, the heat treatment preferably includes the following steps: incubating the ssRNA molecular weight marker at 65-70° C. for 8-10 min, and then cooling it at 0-2° C. for 3-5 min to obtain the heat treatment product.

[0080] Embodiments of the present invention are described in detail below, and the examples of the embodiments are intended to be used to explain the present invention, but should not be construed as limiting the present invention. In the embodiments, if specific techniques or conditions are not specified, the techniques or conditions described in the literature in this area or the product specifications are used. If the manufacturer is not specified for reagents or instruments used, they are all conventional products that can be obtained commercially.

[0081] The reagents and sources involved in the following examples are specifically:

[0082] Fast PCR polymerase Max DNA Polymerase (Takara, catalog number R045);

[0083] Column purification kit Gel DNA Extraction Mini Kit (Novozymes, catalog number DC301);

[0084] DNA marker (Takara, catalog number 3427);

[0085] 10×T7 Transcription Buffer (Novozymes, catalog number DD4101);

[0086] T7 RNA Polymerase (Novozymes, catalog number DD4101);

[0087] Inorganic Pyrophosphatase (Novozyme, catalog number DD4103);

[0088] RNase Inhibitor (Novozyme, catalog number DD4117);

[0089] CIMmultus TM Oligo dT (Sartorius, catalog number 924.1218-2);

[0090] 2X RNA Loading Dye (thermo, catalog number R0641);

[0091] RNA marker (thermo, catalog number SM1823).

[0092] Example

[0093] This example is used to illustrate a method for preparing a ssRNA molecular weight marker, which specifically includes:

[0094] 1. Preparation of DNA template: (1) Gene synthesis was used to obtain 8 DNA template fragments, which were connected to the pET24a plasmid and then polymerized using fast PCR polymerase. Max DNA Polymerase was used to perform PCR on the above 8 plasmids according to the instructions to obtain PCR products.

[0095] PCR reaction system: 10 μM forward primer, 10 μM reverse primer, 1 ng plasmid template, 25 μL fast PCR polymerase Max DNA Polymerase, ddH2O to a total volume of 50 μL. PCR reaction conditions: 95℃3min; 98℃30s, 64℃5s, 72℃70s, 35 cycles; 72℃5min. The primers used for PCR amplification of each plasmid template and the obtained product information are shown in Table 1.

[0096] Table 1.

[0097]

[0098] (2) Using a column purification kit Gel DNA Extraction Mini Kit and purify the PCR products according to the instructions to obtain the DNA template of each ssRNA - dsDNA.

[0099] The purified DNA template was subjected to agarose gel electrophoresis. Figure 1 As shown. Figure 1 The results shown show that the amplified product obtained by PCR using the above plasmid template and primers has a single band and the fragment size is correct.

[0100] 2. Preparation of ssRNA: (1) According to the reagent addition amount and sample addition order shown in Table 2, take each raw material and mix them, pipette and mix them evenly, centrifuge briefly for 2 seconds, and collect the sample at the bottom of the tube. Place each tube in a metal bath and incubate at 37°C for 4 hours to obtain the IVT stock solution.

[0101] Table 2.

[0102] Sample loading order Component name Add volume 1 10×T7 Transcription Buffer 2μL 2 ATP(100mM) 1.5μL 3 CTP (100 mM) 1.5μL 4 GTP (100 mM) 1.5μL 5 UTP(100mM) 1.5μL 6 T7 RNA Polymerase (50U / μL) 2μL 7 Inorganic Pyrophosphatase(0.1U / μL) 1μL 8 RNase Inhibitor (40U / uL) 1μL 9 Nuclease-free water Make up the final volume to 20 μL 10 dsDNA template 1 μg

[0103] Take the IVT stock solution and perform agarose gel electrophoresis. The results are as follows: Figure 2 As shown. Figure 2As shown in the structure, the ssRNA bands in the obtained IVT stock solution are bright, clear, and sharp, and there is no obvious degradation.

[0104] (2) Dilute the IVT stock solution with Buffer 1 (50 mM Tris + 5 mM EDTA + 500 mM NaCl, pH = 7.0) to a volume of 0.55 mL. Take 0.5 mL each time and add it to the CIMmultus TM In Oligo dT, the column was washed with Buffer 1 and then with DEPC water, and the eluate was collected. The eluate was ultrafiltered, desalted and concentrated using a 10Kd ultrafiltration centrifuge tube and DEPC water as a washing liquid to obtain ssRNA. The nucleotide sequences of each ssRNA are shown in Table 3.

[0105] Table 3.

[0106] name SEQ ID NO: Length (nt) ssRNA6000 fragment 25 6000 ssRNA4000 fragment 26 4000 ssRNA3000 fragment 27 3000 ssRNA2000 fragment 28 2000 ssRNA1500 fragment 29 1500 ssRNA1000 fragment 30 1000 ssRNA500 fragment 31 500 ssRNA200 fragment 32 200

[0107] 3. Preparation of ssRNA molecular weight markers: The ssRNA prepared as described above was mixed at 100 ng / band to obtain ssRNA molecular weight markers of 200 to 6000 nt.

[0108] 100 ng and 150 ng of ssRNA molecular weight markers were mixed with 4 μL of 2X RNA Loading Dye (Thermo) and subjected to heat treatment (70°C, 10 min, 5 min on ice) before non-denaturing agarose gel electrophoresis. 100 ng of commercially available RNA marker or 150 ng of commercially available RNA marker subjected to the above heat treatment was used as a control. The results are shown in FIG. Figure 3 As shown. Figure 3 The results shown show that, compared with commercially available RNA markers, the bands in the ssRNA molecular weight marker provided in this example are bright and clear.

[0109] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. A method for preparing a ssRNA molecular weight marker, characterized in that: The preparation method comprises: taking a primer composition to perform PCR amplification on a plasmid template to obtain a DNA template; taking the DNA template to perform in vitro transcription under the action of T7 RNA polymerase to obtain an IVT stock solution; taking the IVT stock solution to perform oligo (dT) enrichment treatment to obtain the ssRNA molecular weight marker; The primer composition comprises a forward primer and a reverse primer, the forward primer comprises a T7 promoter fragment, and the reverse primer comprises a polythymine fragment with a length of 20 to 150 nt.

2. The method for preparing the ssRNA molecular weight marker according to claim 1, characterized in that: The nucleotide sequence of the T7 promoter fragment is specifically: TAATACGACTCACTATA.

3. The method for preparing the ssRNA molecular weight marker according to claim 1, characterized in that: When the plasmid template includes a nucleotide fragment with a sequence as described in SEQ ID NO: 1, the primer composition includes a forward primer with a sequence as described in SEQ ID NO: 2 and a reverse primer with a sequence as described in SEQ ID NO: 3, and the length of the ssRNA molecular weight marker is 6000 nt; Optionally, when the plasmid template includes a nucleotide fragment with a sequence as shown in SEQ ID NO:4, the primer composition includes a forward primer with a sequence as shown in SEQ ID NO:5 and a reverse primer as shown in SEQ ID NO:6, and the length of the ssRNA molecular weight marker is 4000 nt; Optionally, when the plasmid template includes a nucleotide fragment with a sequence as shown in SEQ ID NO:7, the primer composition includes a forward primer with a sequence as shown in SEQ ID NO:8 and a reverse primer as shown in SEQ ID NO:9, and the length of the ssRNA molecular weight marker is 3000 nt; Optionally, when the plasmid template includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 10, the primer composition includes a forward primer with a sequence as shown in SEQ ID NO: 11 and a reverse primer as shown in SEQ ID NO: 12, and the length of the ssRNA molecular weight marker is 2000 nt; Optionally, when the plasmid template includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 13, the primer composition includes a forward primer with a sequence as shown in SEQ ID NO: 14 and a reverse primer as shown in SEQ ID NO: 15, and the length of the ssRNA molecular weight marker is 1500 nt; Optionally, when the plasmid template includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 16, the primer composition includes a forward primer with a sequence as shown in SEQ ID NO: 17 and a reverse primer as shown in SEQ ID NO: 18, and the length of the ssRNA molecular weight marker is 1000 nt; Optionally, when the plasmid template includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 19, the primer composition includes a forward primer with a sequence as shown in SEQ ID NO: 20 and a reverse primer as shown in SEQ ID NO: 21, and the length of the ssRNA molecular weight marker is 500 nt; Optionally, when the plasmid template comprises a nucleotide fragment having a sequence as shown in SEQ ID NO: 22, the primer composition comprises a forward primer having a sequence as shown in SEQ ID NO: 23 and a reverse primer as shown in SEQ ID NO: 24, and the length of the ssRNA molecular weight marker is 200 nt.

4. The method for preparing the ssRNA molecular weight marker according to claim 1, characterized in that: The PCR amplification reaction system includes: Taq enzyme, PCR buffer, dNTP, forward primer, reverse primer and plasmid template; Optionally, the reaction conditions of the PCR amplification include: pre-denaturation at 92-98°C for 1-5 min; denaturation at 92-98°C for 25-35 s, annealing at 60-65°C for 3-10 s, extension at 70-75°C for 65-75 s, 30-40 cycles; 70-75°C for 3-7 min.

5. The method for preparing the ssRNA molecular weight marker according to claim 1, characterized in that: The in vitro transcription reaction system comprises: RNA polymerase, inorganic pyrophosphatase, RNase inhibitor, dNTP, transcription buffer and DNA template; Optionally, the temperature of the in vitro transcription is 35-40° C., and the time is 1-10 h.

6. The method for preparing the ssRNA molecular weight marker according to claim 1, characterized in that: The oligo(dT) enrichment treatment includes one or more of oligo(dT) chromatography enrichment treatment, oligo(dT) magnetic bead enrichment treatment and oligo(dT) centrifugal column enrichment treatment.

7. A ssRNA molecular weight marker, characterized in that The ssRNA molecular weight marker is prepared by the method for preparing the ssRNA molecular weight marker according to any one of claims 1 to 6.

8. The ssRNA molecular weight marker according to claim 7, characterized in that The ssRNA molecular weight markers include one or more of a ssRNA6000 fragment, a ssRNA4000 fragment, a ssRNA3000 fragment, a ssRNA2000 fragment, a ssRNA1500 fragment, a ssRNA1000 fragment, a ssRNA500 fragment, and a ssRNA200 fragment; Among them, the nucleotide sequence of the ssRNA6000 fragment is shown in SEQ ID NO:25, the nucleotide sequence of the ssRNA4000 fragment is shown in SEQ ID NO:26, the nucleotide sequence of the ssRNA3000 fragment is shown in SEQ ID NO:27, the nucleotide sequence of the ssRNA2000 fragment is shown in SEQ ID NO:28, the nucleotide sequence of the ssRNA1500 fragment is shown in SEQ ID NO:29, the nucleotide sequence of the ssRNA1000 fragment is shown in SEQ ID NO:30, the nucleotide sequence of the ssRNA500 fragment is shown in SEQ ID NO:31, and the nucleotide sequence of the ssRNA200 fragment is shown in SEQ ID NO:

32.

9. Use of the ssRNA molecular weight marker according to claim 7 or 8 in molecular detection.

10. Use of the ssRNA molecular weight marker according to claim 9 in molecular detection, characterized in that: The application includes: taking the ssRNA molecular weight marker and performing heat treatment to obtain a heat treatment product; taking the heat treatment product as an RNA marker to determine the molecular weight of a sample to be tested; Optionally, the heat treatment comprises: incubating the ssRNA molecular weight marker at 65-70° C. for 8-10 minutes, and then cooling at 0-2° C. for 3-5 minutes to obtain the heat treatment product.

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